Project Summary: The mechanisms underlying neurodegenerative disease-related psychiatric disorders are unknown. Symptoms of anxiety and depression are shared over many age-related diseases including stroke, Alzheimer?s and Parkinson?s. The critical gap in our understanding of mechanisms driving anxiety and depression now affects the mental health of individuals suffering from neurodegenerative diseases. One brain region thought to be associated with anxiety and depression is the locus coeruleus. The long-term objective of this proposal is to identify the projecting regions/neurons to the locus coeruleus that contributes to anxiety and depression. To achieve this goal, we have constructed specific aims that will allow us to identify the innervating neurons from the amygdala and raphe nucleus to the locus coeruleus, which will give us the ability to modulate only those locus coeruleus neurons. Understanding the specific neural circuits holds great promise for unlocking new therapies for psychiatric disorders. This proposal uses the integration of both established and novel techniques to understand the neuron-to-behavioral circuit and is directly related to the NIMH?s mission statement, which is to transform the understanding and treatment of mental illnesses through basic and clinical research. Therefore, we propose a method for interrogating the neural circuits suspected in anxiety and depression. We have developed a method; we termed innervating cell type Translating Ribosomal Affinity Purification (iTRAP), allowing us to molecularly characterized cell types based on either their anatomic position or monosynaptic connectivity with a defined cell population. Using iTRAP in combination with several novel recombinant rabies viruses (RbV), we can transduce innervating neurons in a retrograde manner. One of these rabies viruses encodes the fusion protein EGFP-ribosomal L10a (EGFPL10a). This allows for the affinity purification of mRNA coupled with RNA sequencing, which provides a molecular signature for cells projecting to the rabies virus injection site. The method further utilizes the TVA-tag strategy to limit the transmission of the RbV-EGFPL10a to monosynaptic inputs of a genetically defined group of cells. Additionally, with the employment of Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) and their activation with a pseudotyped EnvA- RbV-Cre, we can assess the behavioral contribution of the locus coeruleus on downstream regions. With these tools, we will be one step closer to dissecting out the circuits driving anxiety and depression.